Abstract Cytochrome b5 (b5) profoundly influences the catalytic efficiency of many cytochrome P450-catalyzed reactions, yet the mechanism(s) of this action of b5 on P450 enzymes are controversial. Among these b5- regulated activities is the 17,20-lyase activity of P450 17A1 (CYP17A1, steroid 17-hydroxylase/17,20-lyase), which is a key step in the biosynthesis of androgens. Diseases of androgen excess and androgen dependence, including polycystic ovary syndrome and prostate cancer, are extremely common, and the P450 17A1 inhibitor abiraterone acetate (AA) is used to treat prostate cancer, proving the relevance of P450 17A1 in human disease. By indiscriminately inhibiting the 17-hydroxylase activity of P450 17A1 in addition to its 17,20-lyase activity, however, AA causes hypertension and potassium loss unless a potent glucocorticoid is co-administered. Consequently, an unmet clinical need is a selective inhibitor of the 17,20-lyase reaction, which will safely lower testosterone production. We hypothesize that a drug, which disrupts the interaction of b5 with P450 17A1, will selectively block the 17,20-lyase reaction and lower testosterone production without disturbing drug metabolism or requiring chronic glucocorticoid therapy. Our long-term goal is to elucidate the biochemical and physical properties of the b5-P450 17A1 complex that enhance the 17,20-lyase reaction. Our central hypothesis is that b5 binding restricts the dynamics of P450 17A1 and bound substrate, which reduces uncoupling of the 17,20-lyase reaction. Consequently, the objectives of this renewal application are to define the biochemical and biophysical nature of the b5-P450 17A1 interaction, determine the rate-limiting step(s) of the 17,20-lyase reaction, and to probe allosteric sites on the complex. In Aim 1, we will probe the conformational dynamics of P450 17A1 and the changes induced upon binding of substrate and/or b5 using hydrogen-deuterium exchange and mass spectrometry. In Aim 2, with the Scott laboratory, we will use site-directed mutagenesis, rigorous enzymology studies, and x-ray crystallography to probe the functional properties of a second non-active-site steroid-binding site on P450 17A1 and its influence on the individual steps of the 17,20-lyase reaction. In Aim 3, with the Waskell laboratory, we will employ pre- steady state kinetic experiments to dissect the rates of individual steps in the catalytic cycle in the presence and absence of b5. In this manner, we will systematically define the mechanism of action of b5 on the 17,20-lyase activity of P450 17A1 and pave the way for development of better drugs to safely inhibit androgen (and estrogen) production for the treatment of human diseases.